Abstract Surprisingly, platelets are capable of bidirectional interactions with their microenvironment through basic cellular processes that are largely unexplored. In this R35 proposal, we expand on our active research themes: one unraveling the mechanistic role of exocytosis in hemostasis and the other investigating endocytosis as an entry pathway to define the role of platelets in innate immunity. By linking these two areas, we can gain deeper insights into how platelet interact with their microenvironments. Despite advances in understanding signaling from vascular damage detection, our view of how activated platelets execute the steps needed for clot formation is limited. We have probed the mechanisms of platelet secretion and how it affects hemostasis, using genetically altered models, and determined that modulating secretion controls thrombus growth without compromising hemostasis. To build on that advance, a better understanding of platelet exocytosis is clearly needed so logical therapeutic strategies can be developed. Our work on platelet endocytosis, endo-lysosomal trafficking, and processing of endocytosed cargo led to the discovery that platelets take up pathogens, e.g., viruses, and are activated. Increasingly, platelets are being associated with immune responses, yet the mechanisms underlying these non-hemostatic functions are largely unknown. Very little is known about platelet endocytosis and next to nothing is known about how platelets traffic and process endocytosed material. Our R35 research program seeks to fill these gaps in knowledge by taking a holistic approach to the study of platelet ?cell biology?. Building on our innovative past work (>50 publications), we will further define platelet membrane trafficking (endocytosis, exocytosis, cargo sorting/processing, etc.). We hypothesize that bidirectional trafficking processes, endo- and exocytosis, are essential for platelet-specific functions, specifically thrombosis and innate immune responses. To address this hypothesis, we will examine platelet exocytosis and endocytosis at mechanistic and physiological levels using an extensive suite of reagents, transgenic mouse strains, and technologies. Going forward, we will use these powerful tools and approaches to define how platelet membrane trafficking (both exo- and endocytosis) affects hemostasis/thrombosis and immune responses at molecular and organismal levels. The data generated are directly applicable to the understanding and treatment of human disease, especially thrombotic diseases which accounts for 1 in 4 deaths world-wide and chronic viremia, e.g., AIDS/HIV1, which increases CVD risk.